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CD1d Tetramer
Natural killer T cells (NKT) comprise an unusual T cell subset that exhibit properties of both the innate an adaptive immune systems, and have been demonstrated to play a role in bacterial and viral infections. These cells express invariant T cell receptors (Va14/Ja18 in mice, Va24/Ja18 in humans) that only recognize the cluster of differentiation molecule CD1d, an MHC-like molecule expressed on antigen presenting cells (APCs) (1 ). Thus, NKT cells are "CD1d restricted" (Figure 1). CD1d molecules only present lipid antigen to the TCR of T cells. Due to the specificity with which NKT cells recognize only CD1d loaded antigens, antibodies against CD1d can be used to locate NKT cell populations both in vivo and'' in vitro''. Background NKT cells comprise different proportions of the total T cell population on a tissue specific basis. They represent 0.5% of T cells in the periphery, 2% of T cells in the spleen, and up to 30% of the T cells in the liver (2 ). Due to the difference in abundance, NKT cells have been traditionallly difficult to find and characterize. NKT cells are activated by a specific lipid called alpha-galactosylceramide (aGalCer), a lipid originally found in sea sponges. Now made synthetically, this compound is a potent activator of all NKT cells (99-100%). Therefore, NKT cells can be activated by loading aGalCer onto CD1d molecules. The specificity of aGalCer activation and CD1d restriction make it possibly to engineer CD1d tetramer, a molecule made from 4 molecules of soluble CD1d loaded with aGalCer and then biotinylated. Those molecules are then coupled with a linker protein to PE-conjugated strepavidin through the use of BirA catalyzed biotinylation. The resulting molecule is a PE-conjugated CD1d molecule loaded with aGalCer that will specifically bind to the TCR receptor on NKT cells. The bound NKT/CD1d tetramer can then be analyzed on a flow cytometer and the population of NKT cells quantified (3). Expression and Purification of Soluble CD1d Tetramers pRmHa-3 fly vector.jpg|Figure 2: RmHa-3 Vector into which soluble CD1d was cloned. Picture adapted from BIOSS toolbox (7) |link=http://www.bioss.uni-freiburg.de/toolbox/products.php?PL-46 Purification of Soluble CD1d.jpg|Figure 3: Purification of Soluble CD1d/B2m dimers. Picture adapted from Sidobre et al. (6). Production of Tetramers.jpg|Figure 4: Production of CD1d Tetramers. Picture adapted from Sidobre et al (6). Creation of CD1d tetramers were first described by Benlagha et al (4 ) and Matsuda et al. (5 ). These results were summarized in a review by Sidobre and Kronenberg (3 ). Expression of soluble CD1d was performed in a drosophila vector, pD(pRmHa-3) (6 ) because this expression system has been used for other MHC class molecules. In addition, insect cells are eukaryotic, allowing for the expression of this protein. The pD(pRmHa-3) vector has a drosophila metallothionein promoter. Drosophila S2 (Schneider 2) cells were then transfected with the expression vector via calcium phosphate precipitation. After selection, soluble CD1d was purified from the cells using Nickel chromatography. (6) CD1d was then biotinylated through the use of BirA protein and loaded with aGalCer. The biotinylated CD1d/aGalCer molecule was then incubated with PE-conjugated strepavidin to facilitate strong affinity binding of the CD1d/aGalCer molecule to the PE fluorophore. CD1d tetramer is now formed. References 1. Rossjohn, Jamie'' et al''. "Recognition of CD1d-restricted antigens by natural killer T cells." Nature Reviews Immunology.(2012).'' 12(845-857). PMID: 23154222 2. Bendelac, Albert ''et al. "The Biology of NKT cells." Annual Reviews in Immunology. (2007). 25:297-336. PMID:17150027 3. Sidobre and Kronenberg. "CD1 tetramers: a powerful tool for the analysis of glycolipid-reactive T cells." (2002). Volume 268, Issue 1, 107-121. PMID: 12213347 4. Benlagha et al. "In Vivo Identification of Glycolipid Antigen-Specific T cells Using Fluorescent CD1d Tetramers". JEM. (1999) vol. 191 no. 11 1895-1904 PMID: 10839805 5. Matsuda et al. "Tracking the response of natural killer T cells to a glycolipid antigen using CD1d tetramers." J Exp Med. (2000) 192(5):741-54 PMID: 10974039 6.Bioss Toolbox."pD alias pRmHa-3". http://www.bioss.uni-freiburg.de/toolbox/products.php?PL-46